The two H2A histone species H2A.X and H2A.Z have been conserved as separate H2 species throughout evolution, but how the function of these two proteins differs from that of the other H2A species is unknown. The following studies are in progress this year to elucidate the different roles of the H2A species. (l) We are developing procedures for expressing the three human H2A histones in bacteria. The purposes of these studies are to prepare enough H2A.X and H2A.Z to use in various structural and functional assays. Preliminary results suggest that H2A.Z has a different affinity for forming the H2A-H2B dimer, the first level of H2A interaction. X-ray crystallographic studies of the octamer with H2A.X an H2A.Z replacing H2A.1 are planned in collaboration with Prof. Moudrianakis. (2) We are expressing the three human H2A in yeast to see if any of the three can replace the yeast H2A in its chromosomes. These studies are in collaboration with Dr. Randall Morse. (3) We are expressing an altered H2A protein in Chinese Hamster cells and Hela cells in sufficient amounts to be visible by Coomassie Blue stain on histone gels. The purpose of this study is to determine whether histone proteins with altere sequences can be incorporated into mammalian cellular chromosomes. These studies offer the possibility of specifically tagging histones in mammalian cells. One possibility is to tag a histone with a monoclonal antibody epitope; this may enable specific subpopulations of nucleosomes to be isolated by affinity methods. (4) We are studying the specific Ca++-activated phosphorylation of H2A.X or a H2A.X-like protein in mammalian cell nuclei. The phosphorylated protein is either a low-abundance H2A or a phosphorylated derivative of H2A.X with an altere mobility. (5) In collaboration with Dr. Theodore Breitman, we are investigating the effects of trichostatin, a histone deacetylase inhibitor on the acetylation of histones in conjunction with the induced differentiation of HL60 and other human tumor lines.